Pestic. Phytomed. (Belgrade), 25(1), 2010, 59-63 UDC: 630*1:632.7:632.937.1:582.288(497.2) Pestic. fitomed. (Beograd), 25(1), 2010, 59-63 Scientific paper * Naučni rad DOI: 10.2298/PIF1001059D

Naturally-Occurring Entomopathogenic Fungi on Three Bark Species (Coleoptera: ) in Bulgaria

Slavimira A. Draganova1, Danail I. Takov2 and Danail D. Doychev3 1Plant Protection Institute, 35 Panajot Volov Str., 2230 Kostinbrod, Bulgaria ([email protected]) 2Institute of Zoology, Bulgarian Academy of Sciences, 1 Tsar Osvoboditel Boulevard, 1000 Sofia, Bulgaria 3University of Forestry, 10 St. Kliment Ohridski Boulevard, 1756 Sofia, Bulgaria Received: December 3, 2009 Accepted: February 2, 2010

SUMMARY Bark (Coleoptera: Curculionidae: Scolytinae) belong to one of the most damag- ing groups of forest and the activity of their natural enemies – pathogens, parasi- toids, parasites or predators suppressing their population density, is of great importance. Biodiversity of entomopathogenic fungi on bark beetles in Bulgaria has been investi- gated sporadically. The aim of this preliminary study was to find, identify and study mor- phological characteristics of fungal entomopathogens naturally-occurring in populations of three curculionid species – Ips sexdentatus Boern, Ips typographus (L.) and autographus (Ratz.). Dead pest adults were found under the bark of Pinus sylvestris and Picea abies trees col- lected from forests in the Maleshevska and Vitosha Mountains. Fungal pathogens were isolated into pure cultures on SDAY (Sabouraud dextrose agar with yeast extract) and were identified based on morphological characteristics both on the host and in a culture. Morphological characteristics of the isolates were studied by phenotypic methods. The fungal isolates obtained from dead adults of Ips sexdentatus, Ips typographus and D. autographus were found to belong to the species Beauveria bassiana (Bals. – Criv.) Vuillemin, Beauveria brongniartii (Saccardo) Petch and Isaria farinosa (Holmsk.) Fries (anamorph Ascomycota, Sordariomycetes: Hypocreales, Cordycipitaceae). Morphological traits of the isolates are described. Keywords: Bark beetles; Ips sexdentatus; Ips typographus; Dryocoetes autographus; Entomopathogenic fungi; Beauveria bassiana; Beauveria brongniartii; Isaria farinosa; Morphological characteristics; Natural occurrence

59 Slavimira A. Draganova et al.

INTRODUCTION of the northern range of Vitosha Mountain, West Bulgaria). Adults of Ips typographus were found under Ips typographus (L.) and Ips acuminatus Gyll. are the bark of dead spruce trees (Picea abies L.) in 2006. some of the most damaging bark beetles (Coleoptera: Adults of Ips sexdentatus were collected from the bark Curculionidae: Scolytinae) on living trees in Europe of dead Scots pine trees (Pinus sylvestris L.) in 2008 according to Gregoire and Evans (2004). from a second stand that was located at an altitude In Bulgaria, about 300 ha of spruce stands in the fo- of 1230 m above sea level on Maleshevska Mountain rest reserve Bistrishko branishte (Vitosha Mountain) (South-West Bulgaria). were destroyed some years ago due to damages caused Adults of D. autographus were found in 2008 under by Ips typographus (Georgiev, 2006). The activity of the bark of fallen and dead spruce trees in the forest re- their various natural enemies – pathogens, parasitoids, serve of Bistrishko Branishte (Vitosha Mountain). The parasites or predators suppressing their population den- stand was at 1550 m above sea level. sity is of great importance. Dead pest adults were examined for fungal pathogens While Beauveria bassiana (Bals.-Criv.) Vuillemin has in 2008 and 2009 at the Department of Biological and been reported as a fungal pathogen frequently occurring Integrated Pest Control (Plant Protection Institute). in populations of bark beetles, the species Isaria fumo- Cadavers of insects were placed in a moist chamber sorosea Wize (major synonym Paecilomyces fumosorose- to allow fungal reproductive structures to develop. us (Wize) Brown and Smith), Isaria farinosa (Holmsk.) Fungal pathogens were isolated into pure cultures on Fries (major synonym Paecilomyces farinosus (Holmsk. SDAY (Sabouraud dextrose agar with yeast extract) and ex Gray) Brown and Smith), Verticillium lecanii (Zimm.) were identified according to Samson et al. (1988) and Viegas and Metarhizium anisopliae (Metsch.) Sorokin Humber (1997) based on morphological characteris- have been mentioned only in a few reports (Landa et al., tics both on the host and in the culture. Morphological 2001; Jankevica, 2004; Sosnowska et al., 2004). characteristics of the isolates were studied by phenoty- Wegensteiner (2004), reviewing records about path- pic methods. Colonies growing for 14 days at 23oC un- ogens of bark beetles, pointed out that there was in- der daylight were checked on plates with PDA (potato sufficient knowledge of the activity of pathogens on dextrose agar) and SDAY. Conidia and conidiogenous bark beetles worldwide in spite of a presumed poten- cells were observed in smears and durable specimens tial of pathogens in controlling bark beetle popula­ with lactophenol and aniline blue using a transmission tions. According to him, a number of investigations interference microscope BX60 DIC Olympus equipped have shown the occurrence of fungi in bark beetle with digital camera and Cell B image capture software field populations. Several fungal species were most fre- with a calibrated Carl Zeiss micrometer. quently mentioned, including B. bassiana and Isaria sp. (synonyms Spicaria, Paecilomyces). Biodiversity of entomopathogenic fungi on bark bee- RESULTS AND DISCUSSION tles in Bulgaria has been sporadically investigated. An isolate obtained from a dead adult of Ips sexdentatus It was established that the examined mycoses on Boer., collected from a natural environment, has been bark beetles were caused predominantly by the species identified by Draganova et al. (2007) as B. bassiana. Beauveria bassiana (Bals.-Criv.) Vuillemin. Two isolates The aim of this study was to find, identify and study (433 and 434 B. bassiana) from Ips typographus, three the morphological characteristics of fungal entomo- isolates (501, 502 and 503 B. bassiana) from Ips sexden- pathogens occurring naturally in populations of three tatus and one (559 B. bassiana) from D. autographus scolytid species – Ips sexdentatus, Ips typographus and were obtained. One isolate each of the species B. brong- Dryocoetes autographus (Ratz.). niartii (Saccardo) Petch (marked 435) and Isaria fari- nosa (Holmsk.) Fries (marked 500) were isolated in- to pure cultures from Ips typographus and Ips sexden- MATERIAL AND METHODS tatus, respectively. The three fungal species are an- amorphs of Ascomycota belonging to Sordariomycetes: Insects were collected from three localities. Hypocreales, Cordycipitaceae. The first stand was located at an altitude of 1400 m Dead insects placed in a moist chamber were cove- above sea level in a forest on Zlatnite Mostove (part red after three days by dense mycelium of the fungus

60 Pestic. fitomed. (Belgrade), 25(1), 2010, 59-63

Table 1. Size of conidia of Beauveria bassiana (Bals.-Criv.) Vuillemin isolates obtained from adults of bark beetles Size of conidia (μm) Fungal isolate Length Width Average size (minimum – maximum) (minimum – maximum) (Length x Width) 433 Beauveria bassiana 1.04-1.91 1.13-1.56 1.34 x 1.32 434 Beauveria bassiana 0.95-1.91 0.95-1.65 1.28 x 1.25 501 Beauveria bassiana 0.95-1.56 0.78-1.56 0.28 x 1.27 502 Beauveria bassiana 0.86-1.72 0.43-1.72 1.16 x 1.02 503 Beauveria bassiana 1.30-1.99 0.69-1.65 1.63 x 1.11 559 Beauveria bassiana 1.56-2.17 1.30-1.82 1.87 x 1.60

Table 2. Colony growth of the isolates of Beauveria bassiana (Bals.-Criv.) Vuillemin, Beauveria brongniartii (Saccardo) Petch and Isaria farinosa (Holmsk.) Fries on PDA (potato dextrose agar) and SDAY (Sabouraud dextrose agar with yeast extract) during 14 days of cultivation at 23oC

Size of fungal colony on plates Size of fungal colony on Fungal isolate with PDA (mm) plates with SDAY (mm) Diameter Height Diameter Height 433 Beauveria bassiana 19.7 thin layer 21.3 3 434 Beauveria bassiana 17.7 thin layer 28.7 4 501 Beauveria bassiana 34.0 thin layer 20.3 2 502 Beauveria bassiana 27.3 thin layer 18.3 2 503 Beauveria bassiana 27.3 thin layer 20.7 2 559 Beauveria bassiana 21.7 thin layer 21.0 2 435 Beauveria brongniartii 20.7 thin layer 22.7 2 500 Isaria farinosa 34.0 thin layer 36.0 6

- white in the cases of B. bassiana and B. brongniartii Conidia of B. bassiana isolates (Table 1) were small- and yellowish white in the case of I. farinosa. Mycelium er in size than conidia of the species reported by Brady started growing out through the inter-segmental mem- (1979a) and by Evlakhova (1974) as 2-3 x 2-2.5 μm and brane of the host. as 2-3 μm in diameter, respectively, but they were sim- Conidiogenous cells of B. bassiana were densely clus- ilar in size to conidia of the isolate 426 B. bassiana tered in whorls, hyaline, smooth and short. In some with dimensions 1.8-2.0 μm in diameter (Draganova smears and durable specimens of all fungal isolates, it et al., 2007). was noticed that conidiogenous cells were branched, On SDAY, B. bassiana isolates formed round raised which made clusters thicker. Conidiogenous cells had colonies with powdery surface, with pigmentation from globose (isolates 433, 434, 503 and 559 B. bassiana) or white to cream, reverse of the colonies with pale cream flask-like base (501, 502 B. bassiana) terminating in pigmentation. Only the isolate 501 B. bassiana released narrow extended denticulate apex. The growing apex pink pigment into the media until the 10th day, but it repeatedly formed conidium and re-grew just below the paled and gradually disappeared and the redish reverse new conidium, giving a distinctly zig-zag appearance of the colonies became pink-tanned. On PDA the col- (rachis) which in some cases reached 12.86 μm (isola- onies were round, flat, like a hyaline film from radial te 501 B. bassiana) or even up to 18.44 μm (isolate 502 growing mycelium, downy in the centre. The colon­ B. bassiana). Conidia were one-celled, hyaline, thin- ies of B. bassiana isolates grew slowly. After 14 days at walled, hydrophobic, globose in shape, very similar in 23oC and exposure to daylight they were between 17.7 size in different isolates – from 1.16 x 1.02 μm (isolate and 34.0 mm in diameter on PDA and between 18.3 502 B. bassiana) to 1.87 x 1.60 μm (isolate 559 B. bas- and 28.7 mm on SDAY (Table 2). siana) (Table 1). Conidiogenous cells of the isolates of Isolate 435 B. brongniartii had morphological traits B. bassiana have been observed in cultures often to be very similar to the morphology of B. bassiana isolates. aggregated into clavate synnemata. The synnemata of Its colonies on PDA were round, flat, like a hyaline the isolate 501 B. bassiana reached 10 mm in length in film from radial growing mycelium, downy in the cen- older cultures with SDAY. tre. Isolate 435 B. brongniartii grew better on SDAY,

61 Slavimira A. Draganova et al.

forming round raised colonies with powdery surface, species I. farinosa (major synonym Paecilomyces farino- with cream pigmentation, and cream-pigmented rever- sus) (Evlakhova, 1974; Brady, 1979c; Humber, 1997). se of the colonies. The size of colonies reached 20.7 mm In conclusion, although the present study was pre- in diameter on PDA and 22.7 mm on SDAY after 14 liminary, the results contribute to our knowledge of days of cultivation at 23oC. Conidiogenous cells were the diversity of entomopathogenic fungi on bark bee- loose, in small groups, with flask-shaped basal part and tles in Bulgaria. long denticulate rachis with conidia formed on the den- ticles. The conidia of isolate 435 were one-celled, hya- line, smooth, ellipsoidal, measuring 1.86 x 1.08 μm. Acknowledgments These dimensions were rather smaller than those re- ported by Brady (1979b) – 2.5-4.5 x 1.5-3 μm. We are especially indebted to the National Science Glare and Inwood (1998) had described morpholog- Fund, Ministry of Education and Science, Bulgaria, for ical and genetic characterisation of Beauveria spp. from its funding provided for project D002-251. New Zealand. They classified the isolates with conidia longer than 3 μm as B. brongniartii, and isolates with shorter and spherical conidia as B. bassiana. References Using a gene-genealogical approach to investigate molecular phylogenetic diversity of Beauveria and sev- Brady, B.L.K.: Beauveria bassiana. CMI Descriptions of eral presumptively related Cordyceps species, Rehner Pathogenic Fungi and Bacteria, Kew, N 602, 1979a. and Buckley (2005) analyzed 86 isolates from diverse Brady, B.L.K.: Beauveria brongniartii. CMI Descriptions of geographic origins, habitats and hosts. They re- Pathogenic Fungi and Bacteria, Kew, N 603, 1979b. ported B. brongniartii isolates (one of them originat- Brady, B.L.K.: Paecilomyces farinosus. CMI Descriptions of ing from Belgium and with insect host belonging to Pathogenic Fungi and Bacteria, Kew, N 613, 1979c. Coleoptera, Rhizophagidae) with smaller dimensions Draganova, S., Takov, D. and Doychev, D.: Bioassays with of conidia (2.1-2.9 x 1.9-2.3 μm). isolates of Beauveria bassiana (Bals.) Vuill. and Paecilomyces The isolate marked 500, obtained from dead adults farinosus (Holm.) Brown and Smith against Ips sexdentatus of Ips sexdentatus, was identified as Isaria farinosa. Boerner and Ips acuminatus Gyll. (Coleoptera: Scolytidae). Its colonies on PDA were not very different from col- Plant Science, 44(1): 24-28, 2007. onies of B. bassiana and B. brongniartii. They were Evlakhova, A.A.: Entomogenous Fungi. Classification, round, flat, like a hyaline film from radial growing Biology, Practical Significance. Nauka Press, Leningrad, mycelium, downy in the centre. In our opinion, PDA Russia, 1974, pp. 1-260. was not suitable for this species. On SDAY, the colo- Georgiev G.: Ips typographus (L.) and drying of the spruce nies of the isolate 500 I. farinosa were round, raised, stands in Vitosha. Bulgarian Forest, 1(5): 8, 2006. with fluffy surface, yellowish to orange pigmenta- Glare, T.R. and Inwood, A.J.: Morphological and genet- tion, the reverse of the colonies of yellowish orange. ic characterisation of Beauveria spp. from New Zealand. The size of the colonies reached 34 mm in diameter Mycological Research, 102: 250-256, 1998. on PDA and 36 mm on SDAY after 14 days of culti- Gregoire, J.C. and Evans, H.F.: Damage and control of vation at 23oC. Conidiophores arise from dense basal BAWBILT organisms, an overview. In: Bark and Wood felt mycelium of the colonies, making them seem gra- Boring Insects in Living Trees in Europe: a synthesis (Lieutier nular. Conidiophores were septate, hyaline, bearing F., Day K.R., Battisti A., Gregoire J.C., Evans H.F., eds.), groups of swollen lateral cells and whorls of conidioge- Springer Publ., 2004, pp. 19-37. nous cells. Conidiogenous cells were flask shaped phi- Humber, R.: Fungi: identification. In: Manual of Techniques alides, smooth-walled, with average dimensions 5.36 in Insect Pathology (Lacey L.A., ed.), Academic Press, San x 1.07 μm, with cylindrical base tapering into a dis­ Diego, USA, 1997, pp. 153-163. tinctly thin neck. Conidia of the isolate were one-ce- Jankevica, L.: Ecological associations between entomopath- lled, hyaline, smooth, lemon-shaped, with average di- ogenic fungi and pest insects recorded in Latvia. Latvijas mensions 1.88-2.14 x 0.79-1.36 μm, borne on the tip Entomologs, 41: 60-65, 2004. of the phialides, forming dry chains. Landa, Z., Hornak, P. and Bursova, E.: Entomopatogenni The morphological traits of the isolate 500 I. fa- houby asociovane s lykozroutem smrkovym were similar to earlier descriptions of the Ips typographus rinosa L. (Coleoptera, Scolytidae) v oblasti NP a CHKO Sumava.

62 Pestic. fitomed. (Belgrade), 25(1), 2010, 59-63

Aktuality Sumavskeho Vyzkumu, srni 2-4, 124-128, 2001. Sosnowska, D., Balazy, S. Prishchepa, L. and Mikulskaya Rehner, S.A. and Buckley, E.: A Beauveria phylogeny N.: Biodiversity of pathogens in the Białowieźa inferred from nuclear ITS and EF1-α sequences: evidence for forest. Journal of Plant Protection Research, 44(4): 313-321, cryptic diversification and links to Cordyceps teleomorphs. 2004. Mycologia, 97(1): 81-98, 2005. Wegensteiner, R.: Pathogens in bark beetles. In: Bark and Samson, R.A., Evans, H.C. and Latge, J.-P.: Atlas of Wood Boring Insects in Living Trees in Europe: a synthesis Entomopathogenic Fungi. Springer – Verlag, London, UK, (Lieutier F., Day K.R., Battisti A., Gregoire J.C., Evans H.F., 1988, pp. 1-187. eds.), Springer Publ., 2004, pp. 291-313.

Entomopatogene gljive koje se u prirodi javljaju kod tri vrste potkornjaka (Coleoptera: Curculionidae) u Bugarskoj

Rezime Potkornjaci (Coleoptera: Curculionidae: Scolytinae) pripadaju jednoj od najštetnijih gru- pa šumskih insekata, te je delovanje njihovih prirodnih neprijatelja, patogena, parazitoida, parazita i predatora od velikog značaja. Biodiverzitet entomopatogenih gljiva koje se javljaju kod potkornjaka u Bugarskoj malo je istraživan. Cilj ovog početnog ispitivanja bio je da se pronađu, odrede i prouče morfološ- ke karakteristike gljiva kao entomopatogena koji se javljaju u prirodnim populacijama tri vr- ste potkornjaka (Curculionidae) – Ips sexdentatus Boern, Ips typographus (L.) i Dryocoetes au- tographus (Ratz.). Uginuli adulti su prikupljeni ispod kore stabala Pinus sylvestris i Picea abies poreklom sa planina Meleševska i Vitoša. Patogene gljive su izolovane kao čiste kulture na podlozi SDAY (Sabouraud dextrose agar with yeast extract) i određene na osnovu morfoloških osobina kako na domaćinu, tako i u kulturi. Morfološke osobine izolata proučavane su fenotipskim metodama. Izolati gljiva dobijeni sa uginulih adulta Ips sexdentatus, Ips typographus i D. autographus pripadaju vrstama Beauveria bassiana (Bals.-Criv.) Vuillemin, Beauveria brongniartii (Saccardo) Petch i Isaria farinosa (Holmsk.) Fries (anamorf Ascomycota, Sordariomycetes: Hypocreales, Cordycipitaceae). Dat je opis morfoloških osobina izolata. Ključne reči: Potkornjaci; Ips sexdentatus; Ips typographus; Dryocoetes autographus; entomo- patogene gljive; Beauveria bassiana; Beauveria brongniartii; Isaria farinosa; morfološke osobi- ne; pojava u prirodi

63